WO2015040402A1 - Procédés, cellules et organismes - Google Patents
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- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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- A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
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Definitions
- the inventors have devised an approach for introducing one or more desired insertions and/or deletions of known sizes into one or more predefined locations in a nucleic acid (e.g., in a cell or organism genome). They developed techniques to do this either in a sequential fashion or by inserting a discrete DNA fragment of defined size into the genome precisely in a predefined location or carrying out a discrete deletion of a defined size at a precise location.
- the technique is based on the observation that DNA single-stranded breaks are preferentially repaired through the HDR pathway, and this reduces the chances of indels (e.g., produced by NHEJ) in the present invention and thus is more efficient than prior art techniques.
- the inventors have also devised new techniques termed sequential endonuclease- mediated homology directed recombination (sEHDR) and sequential Cas-mediated homology directed recombination (sCHDR).
- sEHDR sequential endonuclease- mediated homology directed recombination
- sCHDR sequential Cas-mediated homology directed recombination
- CRISPR/Cas systems which continually undergo reprogramming to direct degradation of complementary sequences present within invading viral or plasmid DNA.
- Short segments of foreign DNA, called spacers are incorporated into the genome between CRISPR repeats, and serve as a 'memory' of past exposures.
- CRISPR spacers are then used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.
- the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system including the CRISPR associated (Cas) protein has been reconstituted in vitro by a number of research groups allowing for the DNA cleavage of almost any DNA template without the caveat of searching for the right restriction enzyme cutter.
- the CRISPR/Cas system also offers a blunt end cleavage creating a dsDNA or, using mutated Cas versions, a selective single strand-specific cleavage (see Cong etal., Wang etal. & Mali etal. cited below).
- CRISPR/Cas-mediated target DNA or genome modification e.g., a Cas9 nuclease
- CRISPR RNA crRNA
- tracrRNA trans-activating crRNA
- pre-crRNA Transcription of the CRISPR array, containing small fragments (20-30 base-pairs) of the encountered (or target) DNA, into pre-crRNA, which undergoes maturation through the hybridisation with tracrRNA via direct repeats of pre-crRNA.
- the hybridisation of the pre-crRNA and tracrRNA known as guide RNA (gRNA or sgRNA), associates l with the Cas nuclease forming a ribonucleoprotein complex, which mediates conversion of pre- crRNA into mature crRNA.
- gRNA or sgRNA guide RNA
- Mature crRNA:tracrRNA duplex directs Cas9 to the DNA target consisting of the protospacer and the requisite protospacer adjacent motif (CRISPR/cas protospacer-adjacent motif; PAM) via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA on the host genome.
- the Cas9 nuclease mediates cleavage of the target DNA upstream of PAM to create a double-stranded break within the protospacer or a strand-specific nick using mutated Cas9 nuclease whereby one DNA strand-specific cleavage motif is mutated (for example, Cas9 nickase contains a D10A substitution) (Cong etal.).
- Csy4 also known as Cas6f
- Cas6f has no sequence homology to Cas9 but the DNA cleavage occurs through a similar mechanism involving the assembly of a Cas- protein-crRNA complex that facilitates target DNA recognition leading to specific DNA cleavage (Haurwitz etal.).
- the CRISPR/Cas system is a superior genome editing system by which mutations can be introduced with relative ease, simply by designing a single guided RNA complementary to the protospacer sequence on the target DNA.
- the dsDNA break induced by an endonuclease such as Cas9
- NHEJ non-homologous end joining mechanism
- indels inserted or deletions
- a first configuration of the present invention provides:- A method of nucleic acid recombination, the method comprising providing dsDNA comprising first and second strands and
- a second configuration of the present invention provides:-
- a method of nucleic acid recombination comprising
- step (c) optionally obtaining the nucleic acid strand modified in step (b) or a progeny nucleic strand comprising the inserted nucleotide sequence.
- a third configuration of the present invention provides:-
- a method of nucleic acid recombination comprising (a) using nucleic acid cleavage to create first and second breaks in a nucleic acid strand, thereby creating 5' and 3' cut ends and a nucleotide sequence between the ends;
- step (c) optionally obtaining the nucleic acid strand modified in step (b) or a progeny nucleic strand comprising the deletion.
- a fourth configuration of the present invention provides:-
- a method of nucleic acid recombination comprising providing dsDNA comprising first and second strands and
- nucleic acid strands of part (a) and part (b) are repaired to produce a deletion of nucleic acid between the cuts.
- a method of sequential endonuclease-mediated homology directed recombination comprising carrying out the method of any preceding configuration a first time and carrying out the method of any preceding configuration a second time.
- serial nucleic acid modifications e.g., genome modifications
- one or more Cas endonucleases e.g., a Cas9 and/or Cys4 is used in a method of sequential Cas- mediated homology directed recombination (sCHDR).
- a method of nucleic acid recombination comprising providing dsDNA comprising first and second strands and (a) using nucleic acid cleavage to create 5' and 3' cut ends in the first strand;
- a method of nucleic acid recombination comprising
- step (c) optionally obtaining the nucleic acid strand modified in step (b) or a progeny nucleic strand comprising the inserted nucleotide sequence.
- insert nucleotide sequence is at least 10 nucleotides long. 5. The method of any preceding sentence, wherein the insert sequence comprises a site specific recombination site.
- a method of nucleic acid recombination comprising
- step (c) optionally obtaining the nucleic acid strand modified in step (b) or a progeny nucleic strand comprising the deletion.
- step (c) is performed by isolating a cell comprising the modified first strand, or by obtaining a non-human vertebrate in which the method has been performed or a progeny thereof.
- step (c) is performed by isolating a cell comprising the modified first strand, or by obtaining a non-human vertebrate in which the method has been performed or a progeny thereof.
- the nucleic acid strand or the first strand is a DNA strand.
- step (b) is performed by carrying out homologous recombination between an incoming nucleic acid comprising first and second homology arms, wherein the homology arms are substantially homologous respectively to a sequence extending 5' from the 5' end and a sequence extending 3' from the 3' end.
- step (b) is performed by carrying out homologous recombination between an incoming nucleic acid comprising an insert nucleotide sequence flanked by the first and second homology arms, wherein the insert nucleotide sequence is inserted between the 5' and 3' ends.
- step (a) The method of any preceding sentence, wherein Cas endonuclease-mediated cleavage is used in step (a); optionally by recognition of a GG or NGG PAM motif.
- step (a) is carried out by cleavage in one single strand of dsDNA.
- step (a) is carried out by combining in a cell the nucleic acid strand, a Cas endonuclease, a crRNA and a tracrRNA (e.g., provided by one or more gRNAs) for targeting the endonuclease to carry out the cleavage, and optionally an insert sequence for homologous recombination with the nucleic acid strand.
- a Cas endonuclease e.g., provided by one or more gRNAs
- step (b) is performed by carrying out homologous recombination with an incoming nucleic acid comprising first and second homology arms, wherein the homology arms are substantially homologous respectively to a sequence extending 5' from the 5' end and a sequence extending 3' from the 3' end, wherein the second homology arm comprises a PAM sequence such that homologous recombination between the second homology arm and the sequence extending 3' from the 3' end produces a sequence comprising a PAM motif in the product of the method.
- a method of sequential endonuclease-mediated homology directed recombination comprising carrying out the method of any preceding sentence (e.g., when according to sentence 1 using a nickase to cut a single strand of dsDNA; or when dependent from sentence 2 or 5 using a nuclease to cut both strands of dsDNA) a first time and a second time, wherein endonuclease-mediated cleavage is used in each step (a); wherein the product of the first time is used for endonuclease-mediated cleavage the second time, whereby either (i) first and second nucleotide sequences are deleted the first time and the second times respectively; (ii) a first nucleotide sequence is deleted the first time and a second nucleotide sequence is inserted the second time; (iii) a first nucleotide sequence is inserted the first time and a second nucleotide sequence is deleted
- step (b) is performed by carrying out homologous recombination between an incoming nucleic acid comprising first and second homology arms, wherein the homology arms are substantially homologous respectively to a sequence extending 5' from the 5' end and a sequence extending 3' from the 3' end, wherein the incoming nucleic acid comprises no sequence between the first and second homology arms such that sequence between the 5' and 3' ends is deleted by homologous recombination; optionally wherein the second arm comprises a PAM motif such that the product of the second time comprises a PAM motif for use in a subsequent Cas endonuclease-mediated method according to any one of sentences 1 to 26.
- step (b) is performed by carrying out homologous recombination between an incoming nucleic acid comprising first and second homology arms, wherein the homology arms are substantially homologous respectively to a sequence extending 5' from the 5' end and a sequence extending 3' from the 3' end, wherein the insert sequence is inserted between the 5' and 3' ends by homologous recombination; optionally wherein the second arm comprises a PAM motif such that the product of the second time comprises a PAM motif for use in a subsequent Cas endonuclease-mediated method according to any one of sentences 1 to 26.
- step (a) is carried out using Cas endonuclease-mediated cleavage and a gRNA comprising a crRNA and a tracrRNA.
- the crRNA has the structure 5'-X-Y-3', wherein X is an RNA nucleotide sequence (optionally at least 5 nucleotides long) and Y is a crRNA sequence comprising a nucleotide motif that hybridises with a motif comprised by the tracrRNA, wherein X is capable of hybridising with a nucleotide sequence extending 5' from the desired site of the 5' cut end.
- the insert sequence is a synthetic sequence; or comprises a sequence encoding all or part of a protein from a species other than the species from which the first cell is derived; or comprises a regulatory element from said first species.
- a cell or a non-human organism whose genome comprises a modification comprising a non-endogenous nucleotide sequence flanked by endogenous nucleotide sequences, wherein the cell or organism is obtainable by the method of any one of sentences 24 to 40 and wherein the non-endogenous sequence is flanked 3' by a Cas PAM motif; wherein the cell is not comprised by a human; and one, more or all of (a) to (d) applies
- the genome is homozygous for the modification; or comprises the modification at one allele and is unmodified by Cas-mediated homologous recombination at the other allele;
- the non-endogenous sequence comprises all or part of a regulatory element or encodes all or part of a protein
- the non-endogenous sequence is at least 20 nucleotides long; (d) the non-endogenous sequence replaces an orthologous or homologous sequence in the genome.
- the cell or organism of sentence 41, wherein the non-endogenous sequence is a human sequence.
- the PAM motif comprises a sequence selected from CCN, TCN, TTC, AWG, CC, NNAGNN, NGGNG GG, NGG, WGG, CWT, CTT and GAA.
- the cell or organism of any one of sentences 41 to 48, wherein the non- endogenous sequence comprises one or more human antibody gene segments, an antibody variable region or an antibody constant region.
- the insert sequence is a human sequence that replaces or supplements an orthologous non-human sequence.
- a vertebrate e.g., mouse or rat
- a method of isolating an antibody that binds a predetermined antigen comprising
- an antibody e.g., and IgG-type antibody expressed by the B lymphocytes.
- a pharmaceutical composition comprising the antibody or antibodies of sentence 52 and a diluent, excipient or carrier.
- a promoter selected from the group consisting of an embryo-specific promoter (e.g., a Nanog promoter, a Pou5fl promoter or a SoxB promoter).
- the cell, animal or blastocyst of any one of sentences 60 to 63, w5erein the Cas endonuclease sequence is flanked 5' and 3' by transposon elements (e.g., inverted piggyBac terminal elements) or site-specific recombination sites (e.g., loxP and/or a mutant lox, e.g., lox2272 or lox511; or frt).
- transposon elements e.g., inverted piggyBac terminal elements
- site-specific recombination sites e.g., loxP and/or a mutant lox, e.g., lox2272 or lox511; or frt.
- 71 The cell, animal or blastocyst of sentence 68, 69 or 70, wherein the gRNA(s) are flanked 5' and 3' by transposon elements (e.g., inverted piggyBac terminal elements) or site- specific recombination sites (e.g., loxP and/or a mutant lox, e.g., lox2272 or lox511; or frt).
- transposon elements e.g., inverted piggyBac terminal elements
- site- specific recombination sites e.g., loxP and/or a mutant lox, e.g., lox2272 or lox511; or frt.
- a method of nucleic acid recombination comprising providing dsDNA comprising first and second strands and
- nucleic acid strands of part (a) and part (b) are repaired to produce a deletion of nucleic acid between the cuts.
- FIG. 1 A Precise DNA I nsertion in a Predefined Location ( Kl ) : gRNA designed against a predefined location can induce DNA nick using Cas9 DIOA nickase 5' of the PAM sequence (shown as solid black box). Alternatively, gRNA can be used together with Cas9 wild-type nuclease to induce double-stranded DNA breaks 5' of the PAM sequence. The addition of a donor oligo or a donor DNA fragment (single or double stranded) with homology around the breakpoint region containing any form of DNA alterations including addition of endogenous or exogenous DNA can be precisely inserted at the breakpoint junction where the DNA is repaired through HDR.
- Figure 1 B Precise DNA I nsertion in a Predefined Location
- Precise DNA I nsertion in a Predefined Locat ion ( Kl ) This figure shows a more detailed description of the mechanism described in Figure 1A.
- sgRNA designed against a predefined location can induce DNA nick using Cas9 DIOA nickase 5' of the PAM sequence (shown as solid black box).
- sgRNA can be used together with Cas9 wild-type nuclease to induce double-stranded DNA breaks 5' of the PAM sequence.
- a donor oligo or a donor DNA fragment (single or double stranded) with homology arms (HA) around the breakpoint region containing any form of DNA alterations including addition of endogenous or exogenous DNA, can be precisely inserted at the breakpoint junction where the DNA is repaired through HDR.
- HA homology arms
- FIG. 2A Precise DNA Deletion ( KO) : gRNAs targeting flanking region of interest can induce two DNA nicks using Cas9 DIOA nickase in predefine locations containing the desired PAM sequences (shown as solid black box). Alternatively, gRNAs can be used with Cas9 wild-type nuclease to induce two DSB flanking the region of interest. Addition of a donor oligo or a donor DNA fragment (single or double stranded) with homology to region 5' of PAM 1 and 3' of PAM 2 sequence will guide DNA repair in a precise manner via HDR.
- FIG. 2B Precise DNA Deletion ( KO) : This figure shows a more detailed description of the mechanism described in Figure 2A. sgRNAs targeting flanking region of interest can induce two DNA nicks using Cas9 D10A nickase in predefine locations containing the desired PAM sequences (shown as solid black box). Note. The PAMs can be located in opposite DNA strands as suppose to the example depicted in the figure where both PAMs are on the same DNA strand.
- sgRNAs can be used with Cas9 wild-type nuclease to induce two DSB flanking the region of interest.
- Addition of a donor oligo or a donor DNA fragment (single or double stranded) with homology to region 5' of PAM 1 and 3' of PAM 2 sequence will guide DNA repair in a precise manner via HDR.
- DNA repair via HDR will reduce the risk of indel formation at the breakpoint junctions and avoid DNA repair through NHFJ and in doing so, it will delete out the region flanked by the PAM sequence and carry out DNA repair in a pre-determined and pre-defined manner.
- FIG. 3A Precise DNA Deletion and I nsert ion ( KO ⁇ Kl ) : gRNAs targeting flanking region of interest can induce two DNA nicks using Cas9 D10A nickase in predefine locations containing the desired PAM sequences (shown as solid black box). Alternatively, gRNAs can be used with Cas9 wild-type nuclease to induce two DSB flanking the region of interest.
- Figure 3B Precise DNA Deletion and I nsertion ( KO ⁇ Kl ) : s
- gRNAs targeting flanking region of interest can induce two DNA nicks using Cas9 D10A nickase in predefine locations containing the desired PAM sequences (shown as solid black box).
- sgRNAs can be used with Cas9 wild-type nuclease to induce two DSB flanking the region of interest.
- FIG. 4A Recycling PAM For Sequential Genome Edit ing ( Deletions) : gRNAs targeting flanking region of interest can induce two DNA nicks using Cas9 D10A nickase in predefine locations containing the desired PAM sequences (shown as solid black box). Alternatively, gRNAs can be used with Cas9 wild-type nuclease to induce two DSB flanking the region of interest. Addition of a donor oligo or a donor DNA fragment (single or double stranded) with homology to region 5' of PAM 2 and 3' of PAM 3 will guide DNA repair in a precise manner via HDR and in doing so, it will delete out the region between PAM 2 and PAM3.
- This deletion will retain PAM 3 and thus acts as a site for carrying out another round of CRISPR/Cas mediated genome editing.
- Another PAM site e.g.. PAM 1
- PAM 3 sequence can be used in conjunction with PAM 3 sequence to carry out another round of deletion as described above.
- PAM recycling approach many rounds of deletions can be performed in a stepwise deletion fashion, where PAM 3 is recycled after each round. This approach can be used also for the stepwise addition of endogenous or exogenous DNA.
- Figure 4B Recycling PAM For Sequential Genome Editing ( Deletions) : This figure shows a more detailed description of the mechanism described in Figure 4B.
- sgRNAs targeting flanking region of interest can induce two DNA nicks using Cas9 D10A nickase in predefine locations containing the desired PAM sequences.
- sgRNAs can be used with Cas9 wild-type nuclease to induce two DSB flanking the region of interest.
- Addition of a donor oligo or a donor DNA fragment (single or double stranded) with homology to region 5' of PAM 1 (clear PAM box) and 3' of PAM 2 (black PAM box) will guide DNA repair in a precise manner via HDR and in doing so, it will delete out the region between PAM 1 and PAM 2.
- PAM sequence together with unique gRNA can be included in the intruding DNA and targeted back into the site of editing.
- PAM 1 sequence for example can be recycled and thus acts as a site for carrying out another round of CRISPR/Cas mediated genome editing.
- Another PAM site eg. PAM 3, grey PAM box
- PAM 3 grey PAM box
- PAM 1 sequence can be used in conjunction with the recycled PAM 1 sequence to carry out another round of editing (i.e. Insertion) as described above.
- PAM recycling approach many rounds of genome editing can be performed in a stepwise fashion, where PAM 1 is recycled after each round. This approach can be used also for the stepwise addition of endogenous or exogenous DNA.
- FIG. 5A CRI SPR/ Cas mediated Lox I nsertion to facilitate RMCE: gRNAs targeting flanking region of interest can induce two DNA nicks using Cas9 D10A nickase in predefine locations containing the desired PAM sequences (shown as solid black box). Alternatively, gRNAs can be used with Cas9 wild-type nuclease to induce two DSB flanking the region of interest.
- RRS recombinase recognition sequence
- loxP loxP
- lox5171 recombinase recognition sequence
- the introduced RRS can be used as a landing pad for inserting any DNA of interest with high efficiency and precisely using recombinase mediated cassette exchange (RMCE).
- RMCE recombinase mediated cassette exchange
- the inserted DNA of interest could contain selection marker such as PGK-Puro flanked by PiggyBac LTR to allow for the initial selection and upon successful integration into DNA of interest, the selection marker can be removed conveniently by expressing hyperPbase transposase.
- Figure 5B CRI SPR/ Cas mediated Lox I nsertion to facilitate RMCE: This figure shows a more detailed description of the mechanism described in Figure 5A. sgRNAs targeting flanking region of interest can induce two DNA nicks using Cas9 D10A nickase in predefine locations containing the desired PAM sequences (shown as solid black box).
- sgRNAs can be used with Cas9 wild-type nuclease to induce two DSB flanking the region of interest.
- Addition of two donor oligos or donor DNA fragments (single or double stranded) with homology to regions 5' and 3' of each PAM sequences where the donor DNA contains recombinase recognition sequence (RRS) such as loxP and lox5171 will guide DNA repair in a precise manner via HDR with the inclusion of these RRS.
- RRS recombinase recognition sequence
- the targeting of the lox sites can be done sequentially or as a pool in a single step process.
- the introduced RRS can be used as a landing pad for inserting any DNA of interest with high efficiency and precisely using recombinase mediated cassette exchange (RMCE).
- RMCE recombinase mediated cassette exchange
- the PAM sequence can be recycled for another round of CRISPR/Cas mediated genome editing for deleting or inserting DNA of interest.
- the inserted DNA of interest could contain selection marker such as PGK-Puro flanked by PiggyBac LTR to allow for the initial selection and upon successful integration into DNA of interest, the selection marker can be removed conveniently by expressing hyperPbase transposase.
- Figure 6A and 6B Genome modify ion to produce transposon-excisable Cas9 and gRNA
- FIG. 6C Single copy Cas9 Expression : A landing pad initially can be targeted into any locus of choice in mouse ES cells or any other eukaryotic cell line.
- the landing pad will typically contain PiggyBac 5' and 3' LTR, selection marker such as neo for example floxed and a gene less promoter such as PGK in the general configuration shown.
- Targeting is done by homologous recombination and clones are selected on G418.
- the next step will involve RMCE to insert Cas9 linked via a T2A sequence to Puro-delta-tk with the option to insert single or multiple guide RNA using the unique restriction sites (RS).
- RS unique restriction sites
- the orientation of the lox sites are positioned in a manner that only once the intruding DNA containing the Cas9 is inserted into the landing pad, the PGK promoter on the landing pad can activate the transcription and thus the expression of the puromycin and via the T2A transcribe and expression Cas9 production.
- a single stable expression of Cas9 can be achieved.
- the entire Cas9 and guide RNA floxed cassette can be excised using PiggyBac transposase (Pbase) and individual clones can be analysed for genome editing resulting from the introduced guide RNA.
- a stable bank cell line expressing Cas9 can be generated from which multiple engineered cell lines can be generated.
- Figure 7 Schematic representing the gRNA position with respect to gene X, the structure of the targeting vector and the oligo pair used for genotyping the resulting targeted clones.
- Figure 8 A gel image show ing the genotyping results following Cas9 nuclease mediated double stranded DNA break and the subsequent DNA targeting.
- the genotyping shows PCR product (880 bp) specific for the 5'targeted homology arm using oligo pair HAP341/HAP334.
- the left hand gels show genotyping data from 96 ES cell clones transfected with gRNA, human Cas9 nuclease and either a circular targeting vector (plate 1) or a linear targeting vector (Plate 2).
- the right hand side gels shows 96 ES cell clones transfected with gRNA and either a circular targeting vector (plate 3) or a linear targeting vector (Plate 4) but with no human Cas9 nuclease. The percentage of the clones correctly targeted is shown for each transfection.
- Figure 9 Schematic showing t he posit ion of t he gRNAs on a gene to allow for a define deletion of the region in between the two gRNA.
- the oligo pair primer 1 and 2 was used to detect ES clones containing the specific 55 bp deletion.
- Figure 1 0 A 3% agarose gel containing PCR products amplified from 96 ES clones transfected w ith gRNA 1 and 2. Primers 1 and 2 was used to amplify around the two gRNA and any clones containing the define deletion can be seen as a smaller PCR product, which are highlighted by an asterix.
- Figure 1 1 PCR genotyping by amplifying the 5' (top gel) and 3 ' ( bottom gel) targeted homology arms w ithin the Rosa26 gene located on chromosome 6. Correctly targeted clones yielding PCR product for both 5' and 3' junctions are marked with an asterix.
- Figure 1 2 Genotyping for the correct insertion of the Cas9 DNA cassette by PCR amplifying the 5' (top gel) and 3' ( bottom gel) arm of the inserted DNA cassette.
- Figure 1 3 PCR genotyping by amplifying the region around the guide RNA and assessing the PCR product for the presence of indels. Larger indels can be seen directly from the gel as they yielded PCR product shorter than the expected WT DNA suggesting significant deletion.
- genomic DNA from mouse AB2.1 was used to size the corresponding WT PCR product.
- the negative control was a no DNA water control.
- Figure 1 4 PCR amplification of the region flanking the guide RNA using DNA extracted from pups follow ing zygote Cas9/ guide mRNA injection for analysing indel formation.
- Lane 14 shows a gross deletion in that mouse and those lanes marked with an asterix indicate these mice contain smaller indels.
- Table 3 Summary of the sequencing data from the 8 mice analysed and the details of the indels detected are shown. The number refers to the frequency of that particular indel identified in the clones analysed and the description of the indels are shown in brackets. DETAI LED DESCRI PTI ON OF THE I NVENTI ON
- nucleic acid modification techniques An example of a technique for nucleic acid modification is the application of the CRISPR/Cas system. This system has been shown thus far to be the most advanced genome editing system available due, inter alia, to its broad application, the relative speed at which genomes can be edited to create mutations and its ease of use. The inventors, however, believed that this technology can be advanced for even broader applications than are apparent from the state of the art.
- the inventors have devised an approach for introducing one or more desired insertions and/or deletions of known sizes into one or more predefined locations in a nucleic acid (e.g., in a cell or organism genome). They developed techniques to do this either in a sequential fashion or by inserting a discrete DNA fragment of defined size into the genome precisely in a predefined location or carrying out a discrete deletion of a defined size at a precise location.
- the technique is based on the observation that DNA single-stranded breaks are preferentially repaired through the HDR pathway, and this reduces the chances of indels (e.g., produced by NHFJ) in the present invention and thus is more efficient than prior art techniques.
- the invention provides:-
- a method of nucleic acid recombination comprising providing double stranded DNA (dsDNA) comprising first and second strands and (a) using nucleic acid cleavage to create 5' and 3' cut ends in the first strand; and
- the method further comprises replicating the modified first strand to produce a progeny dsDNA wherein each strand thereof comprises a copy of the insert nucleotide sequence.
- the method comprises (c) isolating the progeny dsDNA, e.g., by obtaining a cell containing said progeny dsDNA.
- Replication can be effected, for example in a cell.
- steps (a) and (b) are carried out in a cell and the cell is replicated, wherein the machinery of the cell replicates the modified first strand, e.g., to produce a dsDNA progeny in which each strand comprises the modification.
- the modified DNA strand resulting from step (b) is isolated.
- the method is carried out in vitro.
- the method is carried out in a cell or cell population in vitro.
- the method is carried out to modify the genome of a virus.
- the method is carried out in vivo in an organism.
- the organism is a non-human organism.
- it is a plant or an animal or an insect or a bacterium or a yeast.
- the method is practised on a vertebrate (e.g., a human patient or a non- human vertebrate (e.g., a bird, e.g., a chicken) or non-human mammal such as a mouse, a rat or a rabbit).
- the method is a method of cosmetic treatment of a human or a non-therapeutic, non-surgical, nondiagnostic method, e.g., practised on a human or a non-human vertebrate or mammal (e.g., a mouse or a rat).
- a non-therapeutic, non-surgical, nondiagnostic method e.g., practised on a human or a non-human vertebrate or mammal (e.g., a mouse or a rat).
- the invention also provides:-
- a method of nucleic acid recombination comprising
- step (b) using homologous recombination to insert a nucleotide sequence between the ends, wherein the insert sequence comprises a regulatory element or encodes all or part of a protein; and (c) Optionally obtaining the nucleic acid strand modified in step (b) or a progeny nucleic strand comprising the inserted nucleotide sequence, e.g., by obtaining a cell containing said progeny nucleic acid strand.
- the progeny strand is a product of the replication of the strand produced by step (b).
- the progeny strand is, for example, produced by nucleic acid replication in a cell.
- steps (a) and (b) are carried out in a cell and the cell is replicated, wherein the machinery of the cell replicates the modified strand produced in step (b), e.g., to produce a dsDNA progeny in which each strand comprises the modification.
- the single nucleic acid strand is a DNA or RNA strand.
- the regulatory element is a promoter or enhancer.
- the inserted nucleotide sequence is a plant, animal, vertebrate or mammalian sequence, e.g., a human sequence.
- the sequence encodes a complete protein, polypeptide, peptide, domain or a plurality (e.g. one, two or more) of any one of these.
- the inserted sequence confers a resistance property to a cell comprising the modified nucleic acid produced by the method of the invention (e.g., herbicide, viral or bacterial resistance).
- the inserted sequence encodes an interleukin, receptor (e.g., a cell surface receptor), growth factor, hormone, antibody (or variable domain or binding site thereof), antagonist, agonist; e.g., a human version of any of these.
- the inserted sequence is an exon.
- the inserted nucleotide sequence replaces an orthologous or homologous sequence of the strand (e.g., the insert is a human sequence that replaces a plant, human or mouse sequence).
- the method is carried out in a mouse or mouse cell (such as an ES cell) and the insert replaces an orthologous or homologous mouse sequence (e.g., a mouse biological target protein implicated in disease).
- the method is carried out (e.g., in vitro) in a human cell and the insert replaces an orthologous or homologous human sequence (e.g., a human biological target protein implicated in disease, e.g., a mutated form of a sequence is replaced with a different (e.g., wild-type) human sequence, which may be useful for correcting a gene defect in the cell.
- the cell may be a human ES or iPS or totipotent or pluripotent stem cell and may be subsequently introduced into a human patient in a method of gene therapy to treat and/or prevent a medical disease or condition in the patient).
- the inserted nucleotide sequence is at least 10 nucleotides long, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800 or 900 nucleotides, or at least 1, 2, 3, 5, 10, 20, 50 or lOOkb long.
- the insert sequence comprises a site specific recombination site, e.g., a lox, frt or rox site.
- the site can be a loxP, lox511 or lox2272 site.
- a method of nucleic acid recombination comprising
- step (c) optionally obtaining the nucleic acid strand modified in step (b) or a progeny nucleic strand comprising the deletion.
- the progeny strand is a product of the replication of the strand produced by step (b).
- the progeny strand is, for example, produced by nucleic acid replication in a cell.
- steps (a) and (b) are carried out in a cell and the cell is replicated, wherein the machinery of the cell replicates the modified strand produced in step (b), e.g., to produce a dsDNA progeny in which each strand comprises the modification.
- the single nucleic acid strand is a DNA or RNA strand.
- the deleted sequence comprises a regulatory element or encodes all or part of a protein.
- the deleted regulatory element is a promoter or enhancer.
- the deleted nucleotide sequence is a plant, animal, vertebrate or mammalian sequence, e.g., a human sequence.
- the sequence encodes a complete protein, polypeptide, peptide, domain or a plurality (e.g. one, two or more) of any one of these.
- the deleted sequence encodes an interleukin, receptor (e.g., a cell surface receptor), growth factor, hormone, antibody (or variable domain or binding site thereof), antagonist, agonist; e.g., a non- human version of any of these.
- the deleted sequence is an exon.
- the deleted nucleotide sequence is replaced by an orthologous or homologous sequence of a different species or strain (e.g., a human sequence replaces an orthologous or homologous plant, human or mouse sequence).
- a human sequence replaces an orthologous or homologous plant, human or mouse sequence.
- the method is carried out in a mouse or mouse cell and the insert replaces an orthologous or homologous mouse sequence (e.g., a mouse biological target protein implicated in disease).
- the method is carried out (e.g., in vitro) in a human cell and the insert replaces an orthologous or homologous human sequence (e.g., a human biological target protein implicated in disease, e.g., a mutated form of a sequence is replaced with a different (e.g., wild-type) human sequence, which may be useful for correcting a gene defect in the cell.
- the cell may be a human ES or iPS or totipotent or pluripotent stem cell and may be subsequently introduced into a human patient in a method of gene therapy to treat and/or prevent a medical disease or condition in the patient).
- the deleted nucleotide sequence is at least 10 nucleotides long, e.g., at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800 or 900 nucleotides, or at least 1, 2, 3, 5, 10, 20, 50 or lOOkb long.
- step (c) is performed by isolating a cell comprising the modified first strand, or by obtaining a non- human vertebrate in which the method has been performed or a progeny thereof.
- the product of the method comprises a nucleic acid strand comprising a PAM motif 3' of the insertion or deletion.
- the PAM motif is within 10, 9, 8, 7 6, 5, 4 or 3 nucleotides of the insertion or deletion. This is useful to enable serial insertions and/or deletions according to the method as explained further below.
- the product of the method comprises a nucleic acid strand comprising a PAM motif 5' of the insertion or deletion.
- the PAM motif is within 10, 9, 8, 7 6, 5, 4 or 3 nucleotides of the insertion or deletion. This is useful to enable serial insertions and/or deletions according to the method as explained further below.
- step (b) is performed by carrying out homologous recombination between an incoming nucleic acid comprising first and second homology arms, wherein the homology arms are substantially homologous respectively to a sequence extending 5' from the 5' end and a sequence extending 3' from the 3' end.
- the incoming nucleic acid comprises first and second homology arms, and the insert sequence and an optional selection marker sequence (e.g., neo nucleotide sequence).
- the arms may be at least 20, 30, 40, 50, 100 or 150 nucleotides in length, for example. Where deletion is required, the insert is omitted (although an optional selection marker sequence may or may not be included between the arms).
- step (b) is performed by carrying out homologous recombination between an incoming nucleic acid comprising an insert nucleotide sequence flanked by the first and second homology arms, wherein the insert nucleotide sequence is inserted between the 5' and 3' ends.
- the insert is between the homology arms and there is no further sequence between the arms.
- each homology arm is at least 20, 30, 40, 50, 100 or 150 nucleotides long.
- step (a) is carried out using an endonuclease, e.g., a nickase.
- an endonuclease e.g., a nickase.
- Nickases cut in a single strand of dsDNA only.
- the endonuclease is an endonuclease of a CRISPR/Cas system, e.g., a Cas9 or Cys4 endonuclease (e.g., a Cas9 or Cys4 nickase).
- the endonuclease recognises a PAM listed in Table 1 below, for example, the endonuclease is a Cas endonuclease that recognises a PAM selected from CCN, TCN, TTC, AWG, CC, NNAGNN, NGGNG GG, NGG, WGG, CWT, CTT and GAA.
- the Cas endonuclease is a S pyogenes endonuclease, e.g., a S pyogenes Cas9 endonuclease.
- a S. pyogenes PAM sequence or Streptococcus thermophilus LMD-9 PAM sequence is used.
- the endonuclease is a Group 1 Cas endonuclease. In an example, the endonuclease is a Group 2 Cas endonuclease. In an example, the endonuclease is a Group 3 Cas endonuclease. In an example, the endonuclease is a Group 4 Cas endonuclease. In an example, the endonuclease is a Group 7 Cas endonuclease. In an example, the endonuclease is a Group 10 Cas endonuclease.
- the endonuclease recognises a CRISPR/Cas Group 1 PAM. In an example, the endonuclease recognises a CRISPR/Cas Group 2 PAM. In an example, the endonuclease recognises a CRISPR/Cas Group 3 PAM. In an example, the endonuclease recognises a CRISPR/Cas Group 4 PAM. In an example, the endonuclease recognises a CRISPR/Cas Group 7 PAM. In an example, the endonuclease recognises a CRISPR/Cas Group 10 PAM.
- Cas endonuclease-mediated cleavage is used in step (a); optionally by recognition of a GG or NGG PAM motif.
- the first and/or second homology arm comprises a PAM motif. This is useful to enable serial insertions and/or deletions according to the method as explained further below.
- a suitable nickase is S pyogenes Cas9 D10A nickase (see Cong et a/, and the .Examples section below).
- steps (a) and (b) of the method is carried out in a cell, e.g. a bacterial, yeast, eukaryotic cell, plant, animal, mammal, vertebrate, non-human animal, rodent, rat, mouse, rabbit, fish, bird or chicken cell.
- a cell e.g. a bacterial, yeast, eukaryotic cell, plant, animal, mammal, vertebrate, non-human animal, rodent, rat, mouse, rabbit, fish, bird or chicken cell.
- the cell is an E ⁇ x>//cell or CHO or HEK293 or Picchia or Saccharomyces cell.
- the cell is a human cell in vitro.
- the cell is an embryonic stem cell (ES cell, e.g., a human or non-human ES cell, such as a mouse ES cell) or an induced pluripotent stem cell (iPS cell; e.g., a human, rodent, rat or mouse iPS cell) or a pluripotent or totipotent cell.
- ES cell e.g., a human or non-human ES cell, such as a mouse ES cell
- iPS cell induced pluripotent stem cell
- the cell is not an embryonic cell, e.g. wherein the cell is not a human embryonic cell.
- the cell is not a pluripotent or totipotent cell.
- the method is used to produce a human stem cell for human therapy (e.g., an iPS cell generated from a cell of a patient for reintroduction into the patient after the method of the invention has been performed on the cell), wherein the stem cell comprises a nucleotide sequence or gene sequence inserted by the method of the invention.
- a human stem cell for human therapy e.g., an iPS cell generated from a cell of a patient for reintroduction into the patient after the method of the invention has been performed on the cell
- the stem cell comprises a nucleotide sequence or gene sequence inserted by the method of the invention.
- the method is carried out in a mammalian cell.
- the cell is a human cell in vitro or a non-human mammalian cell.
- a non-human (e.g., rodent, rat or mouse) zygote for example, a single-cell non-human zygote.
- the method is carried out in a plant or non-human mammal, e.g. a rodent, mouse or rat or rabbit, or a tissue or organ thereof (e.g., in vitro).
- a plant or non-human mammal e.g. a rodent, mouse or rat or rabbit, or a tissue or organ thereof (e.g., in vitro).
- the 3' or each cleavage site is flanked 3' by PAM motif (e.g., a motif disclosed herein, such as NGG or NGGNG sequence, wherein N is any base and G is a guanine).
- PAM motif e.g., a motif disclosed herein, such as NGG or NGGNG sequence, wherein N is any base and G is a guanine.
- one or more or all cleavage sites are flanked 3' by the sequence 5'-TGGTG-3'.
- the PAM is not absolutely required for ssDNA binding and cleavage: A single- stranded oligodeoxynucleotide containing a protospacer with or without a PAM sequence is bound nearly as well as dsDNA and may be used in the invention wherein a single strand of DNA is modified.
- Cas9 cuts ssDNA bound to the crRNA using its HNH active site independently of PAM.
- step a) is
- (a) is carried out by cleavage in one single strand of dsDNA or in ssDNA.
- step (a) is carried out by combining in a cell the nucleic acid strand, a Cas endonuclease, a crRNA and a tracrRNA (e.g., provided by one or more gRNAs) for targeting the endonuclease to carry out the cleavage, and optionally an insert sequence for homologous recombination with the nucleic acid strand.
- a Cas endonuclease is encoded by a nucleotide sequence that has been introduced into the cell.
- the gRNA is encoded by a DNA sequence that has been introduced into the cell.
- step (b) is performed by carrying out homologous recombination with an incoming nucleic acid comprising first and second homology arms, wherein the homology arms are substantially homologous respectively to a sequence extending 5' from the 5' end and a sequence extending 3' from the 3' end, wherein the second homology arm comprises a PAM sequence such that homologous recombination between the second homology arm and the sequence extending 3' from the 3' end produces a sequence comprising a PAM motif in the product of the method.
- the PAM can be any PAM sequence disclosed herein, for example.
- the method produces a modified nucleic acid strand comprising a PAM that can be used for a subsequent nucleic acid modification according to any configuration, aspect, example or embodiment of the invention, wherein a Cas endonuclease is used to cut the nucleic acid.
- a Cas endonuclease is used to cut the nucleic acid.
- This is useful, for example, for performing sequential endonuclease-mediated homology directed recombination (sEHDR) according to the invention, more particularly sCHDR described below.
- sEHDR sequential endonuclease-mediated homology directed recombination
- the invention further provides:-
- a method of sequential endonuclease-mediated homology directed recombination comprising carrying out the method of any preceding configuration, aspect, example or embodiment of the invention a first time and a second time, wherein endonuclease-mediated cleavage is used in each step (a); wherein the product of the first time is used for endonuclease- mediated cleavage the second time, whereby either (i) first and second nucleotide sequences are deleted the first time and the second times respectively; (ii) a first nucleotide sequence is deleted the first time and a second nucleotide sequence is inserted the second time; (iii) a first nucleotide sequence is inserted the first time and a second nucleotide sequence is deleted the second time; or (iv) first and second nucleotide sequences are inserted the first and second times respectively; optionally wherein the nucleic acid strand modification the second time is within 20, 10, 5, 4, 3, 2
- first and second nucleotide sequences are inserted so that they are contiguous after the insertion the second time.
- first and second deletions are such that a contiguous sequence has been deleted after the first and second deletions have been performed.
- the invention uses a Cas endonuclease.
- a method of sequential Cas-mediated homology directed recombination comprising carrying out the method of any preceding claim a first time and a second time, wherein Cas endonuclease-mediated cleavage is used in each step (a); wherein step (b) of the first time is carried out performing homologous recombination with an incoming nucleic acid comprising first and second homology arms, wherein the homology arms are substantially homologous respectively to a sequence extending 5' from the 5' end and a sequence extending 3' from the 3' end, wherein the second homology arm comprises a PAM sequence such that homologous recombination between the second homology arm and the sequence extending 3' from the 3' end produces a sequence comprising a PAM motif in the product of the method; wherein the PAM motif of the product of the first time is used for Cas endonuclease-mediated cleavage the second time, whereby either (i) first and second nucleotide sequences
- first and second nucleotide sequences are inserted so that they are contiguous after the insertion the second time.
- first and second deletions are such that a contiguous sequence has been deleted after the first and second deletions have been performed.
- the first time is carried out according to the third configuration of the invention, wherein the incoming nucleic acid comprises no sequence between the first and second homology arms, wherein sequence between the 5' and 3' ends is deleted by homologous recombination; and/or the second time is carried out according to the third configuration of the invention, wherein step (b) is performed by carrying out homologous recombination between an incoming nucleic acid comprising first and second homology arms, wherein the homology arms are substantially homologous respectively to a sequence extending 5' from the 5' end and a sequence extending 3' from the 3' end, wherein the incoming nucleic acid comprises no sequence between the first and second homology arms such that sequence between the 5' and 3' ends is deleted by homologous recombination; optionally wherein the second arm comprises a PAM motif such that the product of the second time comprises a PAM motif for use in a subsequent Cas endonuclease-mediated method according to any configuration,
- the first time is carried out according to the first or second configuration of the invention, wherein the incoming nucleic acid comprises the insert sequence between the first and second homology arms, wherein the insert sequence is inserted between the 5' and 3' ends by homologous recombination; and/or the second time is carried out according to the first or second configuration of the invention, wherein step (b) is performed by carrying out homologous recombination between an incoming nucleic acid comprising first and second homology arms, wherein the homology arms are substantially homologous respectively to a sequence extending 5' from the 5' end and a sequence extending 3' from the 3' end, wherein the insert sequence is inserted between the 5' and 3' ends by homologous recombination; optionally wherein the second arm comprises a PAM motif such that the product of the second time comprises a PAM motif for use in a subsequent Cas endonuclease-mediated method according to any configuration, aspect, example or embodiment of
- one of said first and second times is carried out as specified in the First
- Embodiment and the other time is carried out as specified in the Second Embodiment, wherein at least one sequence deletion and at least one sequence insertion is performed.
- step (a) is carried out by Cas endonuclease-mediated cleavage using a Cas endonuclease, one or more crRNAs and a tracrRNA.
- the method is carried out in a cell and the crRNA and tracrRNA is introduced into the cell as RNA molecules.
- the method is carried out in a zygote (e.g., a non-human zygote, e.g., a rodent, rat or mouse zygote) and the crRNA and tracrRNA is injected into zygote.
- the crRNA and tracrRNA are encoded by DNA within a cell or organism and are transcribed inside the cell (e.g., an ES cell, e.g., a non-human ES cell, e.g., a rodent, rat or mouse ES cell) or organism to produce the crRNA and tracrRNA.
- the organism is, for example, a non-human animal or plant or bacterium or yeast or insect.
- the tracrRNA is in this way encoded by DNA but one or more crRNAs are introduced as RNA nucleic acid into the cell or organism to effect the method of the invention.
- the endonuclease may be introduced as a protein or a vector encoding the endonuclease may be introduced into the cell or organism to effect the method of the invention.
- the endonuclease is encoded by DNA that is genomically integrated into the cell or organism and is transcribed and translated inside the cell or organism.
- the method of the invention is carried out in an ES cell (e.g., a non- human ES cell, e.g., a rodent, rat or mouse ES cell) that has been pre-engineered to comprise an expressible genomically-integrated Cas endonuclease sequence (or a vector carrying this has been include in the cell). It would be possible to introduce (or encode) a tracrRNA. By introducing a crRNA with a guiding oligo sequence to target the desired area of the cell genome, one can then carry out modifications in the cell genome as per the invention. In an example, a gRNA as described herein is introduced into the ES cell.
- a gRNA as described herein is introduced into the ES cell.
- the genomically-integrated expressible Cas endonuclease sequence can, for example, be constitutively expressed or inducibly expressible. Alternatively or additionally, the sequence may be expressible in a tissue-specific manner in a progeny organism (e.g., a rodent) developed using the ES cell.
- a progeny organism e.g., a rodent
- the initial ES cell comprising a genomically-integrated expressible Cas endonuclease sequence can be used, via standard techniques, to produce a progeny non-human animal that contains the expressible Cas endonuclease sequence.
- the invention provides:- A non-human animal (e.g., a vertebrate, mammal, fish or bird), animal cell, insect, insect cell, plant or plant cell comprising a genomically-integrated expressible Cas endonuclease nucleotide sequence and optionally a tracrRNA and/or a nucleotide sequence encoding a tracrRNA.
- the Cas endonuclease is, for example, Cas9 or Cys4.
- the animal, insect or plant genome comprises a chromosomal DNA sequence flanked by site-specific recombination sites and/or transposon elements (e.g., piggyBac transposon repeat elements), wherein the sequence encodes the endonuclease and optionally one or more gRNAs.
- transposon elements e.g., piggyBac transposon repeat elements
- the transposon elements can be used to excise the sequence from the genome once the endonuclease has been used to perform recombination.
- the RMCE and/or transposon-mediated excision can be performed in a cell (e.g., an ES cell) that later is used to derive a progeny animal or plant comprising the desired genomic modification.
- the invention also provides an ES cell derived or derivable from such an animal, wherein the ES cell comprises a genomically-integrated expressible Cas endonuclease nucleotide sequence.
- the ES cell is a rodent, e.g., a mouse or rat ES cell, or is a rabbit, dog, pig, cat, cow, non-human primate, fish, amphibian or bird ES cell.
- the invention also provides a method of isolating an ES cell, the method comprising deriving an ES cell from an animal (e.g., a non-human animal, e.g., a rodent, e.g., a rat or a mouse), wherein the animal comprises a genomically-integrated expressible Cas endonuclease nucleotide sequence, as described herein.
- an animal e.g., a non-human animal, e.g., a rodent, e.g., a rat or a mouse
- the animal comprises a genomically-integrated expressible Cas endonuclease nucleotide sequence, as described herein.
- an iPS or stem cell can be derived from (e.g., a somatic cell of) a human, engineered in vitro to comprise a genomically-integrated expressible Cas endonuclease nucleotide sequence and optionally one or more DNA sequences encoding a tracrRNA or gRNA.
- the invention also relates to such a method and to a human iPS or stem cell comprising a genomically-integrated expressible Cas endonuclease nucleotide sequence and optionally one or more DNA sequences encoding a tracrRNA or gRNA.
- This cell can be used in a method of the invention to carry out genome modification (e.g., to correct a genetic defect, e.g., by replacement of defective sequence with a desired sequence, optionally with subsequent transposon-mediated excision of the endonuclease-encoding sequence).
- the iPS cell or stem cell can be introduced into the donor human (or a different human, e.g., a genetic relative thereof) to carry out genetic therapy or prophylaxis.
- a totipotent or pluripotent human cell is used and then subsequently developed into human tissue or an organ or part thereof. This is useful for providing material for human therapy or prophylaxis or for producing assay materials (e.g., for implantation into model non-human animals) or for use in in vitro testing (e.g., of drugs).
- the method uses a single guided RNA (gRNA or sgRNA) comprising a crRNA and a tracrRNA.
- the crRNA comprises an oligonucleotide sequence ("X" in the structure 5'-X-Y-3' mentioned below) that is chosen to target a desired part of the nucleic acid or genome to be modified.
- X oligonucleotide sequence
- the sequence is from 3 to 100 nucleotides long, e.g., from 3 to 50, 40, 30, 25, 20, 15 or 10 nucleotides long, e.g., from or 5, 10, 15 or 20 to 100 nucleotides long, e.g., from 5, 10, 15 or 20 to 50 nucleotides long.
- the gRNA is a single nucleic acid comprising both the crRNA and the tracrRNA.
- An example of a gRNA comprises the sequence 5'-[oligo]-[UUUUAGAGCUA (S N1UUUUAN2N3GCUA)]-[LINKER]-[UAGCAAGUUAAAA (SEQ ID NO:2)]-3', wherein the LINKER comprises a plurality (e.g., 4 or more, e.g., 4, 5 or 6) nucleotides (e.g., 5'-GAAA-3')-
- the crRNA has the structure 5'-X-Y-3', wherein X is an RNA nucleotide sequence (optionally, at least 5 nucleotides long) and Y is a crRNA sequence comprising a nucleotide motif that hybridises with a motif comprised by the tracrRNA, wherein X is capable of hybridising with a nucleotide sequence 5' of the desired site of the 5' cut end, e.g., extending 5' from the desired site of the 5' cut.
- the spacer sequence is, e.g., 5, 4, 3, 2 or 1 RNA nucleotides in length (e.g., AAG in 5' to 3' orientation).
- M2 is, for example, an A, U, C or G (e.g., M2 is a G).
- a chimaeric gRNA is used which comprises a sequence 5'-X-Y-Z- 3', wherein X and Y are as defined above and Z is a tracrRNA comprising the sequence (in 5' to 3' orientation) UAGCM1UUAAAAM2 (SEQ ID NO:4), wherein Ml is spacer nucleotide sequence and M2 is a nucleotide.
- Z comprises the sequence 5'- UAGCAAGUUAAAA-3' (SEQ ID NO:2), e.g., Z is 5'- UAGCAAGUUAAAAUAAGGCUAGUCCG-3' (SEQ ID NO:5).
- the gRNA has the sequence: 5'-GUUUUAGAGCUAGAMUAGCMGUUAA UMGGCUAGUCCGUUAUCMCUUGAAAAAGUGGCA CCGAGUCGGUGC-3' (SEQ ID NO: 6)
- the exogenous sequence can be provided on linear or circular nucleic acid (e.g., DNA).
- the exogenous sequence is flanked by homology arms that can undergo homologous recombination with sequences 5' and 3' respectively of the site where the exogenous sequence is to be inserted.
- homology arms that can undergo homologous recombination with sequences 5' and 3' respectively of the site where the exogenous sequence is to be inserted.
- the skilled person is familiar with choosing homology arms for homologous recombination.
- the invention can be used in a method of producing a transgenic organism, e.g., any organism recited herein.
- the organism can be a non-human organism used as an assay model to test a pharmaceutical drug or to express an exogenous protein or a part thereof (e.g., a human protein target knocked-in into a non-human animal assay organism).
- the invention has been used to knock-out an endogenous sequence (e.g., a target protein) in an organism, such as a non-human organism. This can be useful to assess the effect (phenotype) of the knock-out and thus to assess potential drug targets or proteins implicated in disease.
- the organism is a non-human animal (e.g., a vertebrate, mammal, bird, fish, rodent, mouse, rat or rabbit) in which a human target protein has been knocked-in using the invention.
- the invention has been used to knock out an orthologous or homologous endogenous target of the organism (e.g., an endogenous target sequence has been replaced at the endogenous position by an orthologous or homologous human target sequence).
- an assay model can be produced for testing pharmaceutical drugs that act via the human target.
- the organism is a non-human vertebrate that expresses human antibody variable regions whose genome comprises a replacement of an endogenous target with an orthologous or homologous human sequence.
- the method of the invention is used to produce an Antibody-Generating Vertebrate or Assay Vertebrate as disclosed in WO2013061078, the disclosure of which, and specifically including the disclosure of such Vertebrates, their composition, manufacture and use, is included specifically herein by reference as though herein reproduced in its entirety and for providing basis for claims herein.
- an exogenous regulatory element is knocked-in using the method.
- it is knocked-in to replace an endogenous regulatory element.
- the invention provides a method of producing a cell or a transgenic non-human organism (e.g., any non-human organism recited herein), the method comprising:
- the organism or cell is homozygous for the modification (i) and/or (ii).
- the cell or organism is a rodent (e.g., a mouse or rat) cell or a rabbit, bird, fish, chicken, non-human primate, monkey, pig, dog, Camelid, shark, sheep, cow or cat cell.
- rodent e.g., a mouse or rat
- rabbit, bird, fish, chicken, non-human primate, monkey, pig, dog, Camelid, shark, sheep, cow or cat cell e.g., a mouse or rat
- the target sequence is an endogenous sequence comprising all or part of a regulatory element or encoding all or part of a protein.
- the insert sequence is a synthetic sequence; or comprises a sequence encoding all or part of a protein from a species other than the species from which the first cell is derived; or comprises a regulatory element from said first species. This is useful to combine genes with new regulatory elements.
- the insert sequence encodes all or part of a human protein or a human protein subunit or domain.
- the insert sequence encodes a cell membrane protein, secreted protein, intracellular protein, cytokine, receptor protein (e.g., Fc receptor protein, such as FcRn or a FcY receptor protein), protein of the human immune system or domain thereof (e.g., an Ig protein or domain, such as an antibody or TCR protein or domain, or a MHC protein), a hormone or growth factor.
- Fc receptor protein e.g., Fc receptor protein, such as FcRn or a FcY receptor protein
- protein of the human immune system or domain thereof e.g., an Ig protein or domain, such as an antibody or TCR protein or domain, or a MHC protein
- the invention also provides:-
- a cell e.g., an isolated or purified cell, e.g., a cell in vitro, or any cell disclosed herein
- a non- human organism e.g., any organism disclosed herein, such as a mouse
- the cell or organism is obtainable by the method of any configuration, aspect, example or embodiment of the invention, and wherein the non- endogenous sequence is flanked 3' and/or 5' by (e.g., within 20, 10, 5, 4, 3, 2 or 1 or less nucleotides of, or directly adjacent to) a Cas PAM motif; wherein the cell is not comprised by a human; and one, more or all of (a) to (d) applies (for example, (a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (c); (b
- the non-endogenous sequence comprises all or part of a regulatory element or encodes all or part of a protein
- the non-endogenous sequence is at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800 or 900 nucleotides, or at least 1, 2, 3, 5, 10, 20, 50 or lOOkb long;
- the non-endogenous sequence replaces an orthologous or homologous sequence in the genome.
- the cell can be a human cell, or included in human tissue but not part of a human being.
- the cell is a human cell in vitro.
- the non-endogenous sequence is a human sequence.
- the PAM motif is any PAM disclosed herein or comprises a sequence selected from CCN, TCN, TTC, AWG, CC, NNAGNN, NGGNG GG, NGG, WGG, CWT, CTT and GAA.
- the motif is a Cas9 PAM motif.
- the PAM is NGG. In another example, the PAM is GG.
- PAM motif no more than 10 nucleotides (e.g., 3 nucleotides) 3' and/or 5' of the non-endogenous sequence.
- the PAM motif is recognised by a Streptococcus Cas9.
- the cell or organism is a non-human vertebrate cell or a non-human vertebrate that expresses one or more human antibody heavy chain variable domains (and optionally no heavy chain variable domains of a non-human vertebrate species).
- the organism is an Antibody-Generating Vertebrate or Assay Vertebrate disclosed in WO2013061078.
- the cell or organism is a non-human vertebrate cell or a non-human vertebrate that expresses one or more human antibody kappa light chain variable domains (and optionally no kappa light chain variable domains of a non-human vertebrate species).
- the cell or organism is a non-human vertebrate cell or a non-human vertebrate that expresses one or more human antibody lambda light chain variable domains (and optionally no kappa light chain variable domains of a non-human vertebrate species).
- the non-endogenous sequence encodes a human Fc receptor protein or subunit or domain thereof (e.g., a human FcRn or FcY receptor protein, subunit or domain).
- the non-endogenous sequence comprises one or more human antibody gene segments, an antibody variable region or an antibody constant region.
- the insert sequence is a human sequence that replaces or supplements an orthologous non-human sequence.
- the invention also provides:-
- a monoclonal or polyclonal antibody prepared by immunisation of a vertebrate (e.g., mouse or rat) of the invention (or produced by a method of the invention) with an antigen.
- the invention also provides:- A method of isolating an antibody that binds a predetermined antigen, the method comprising:
- an antibody e.g., an IgG-type antibody expressed by the B lymphocytes.
- the method comprises the step of isolating from said B lymphocytes nucleic acid encoding said antibody that binds said antigen; optionally exchanging the heavy chain constant region nucleotide sequence of the antibody with a nucleotide sequence encoding a human or humanised heavy chain constant region and optionally affinity maturing the variable region of said antibody; and optionally inserting said nucleic acid into an expression vector and optionally a host.
- the method comprises making a mutant or derivative of the antibody produced by the method.
- the invention provides the use of an isolated, monoclonal or polyclonal antibody described herein, or a mutant or derivative antibody thereof that binds said antigen, in the manufacture of a composition for use as a medicament.
- the invention provides the use of an isolated, monoclonal or polyclonal antibody described herein, or a mutant or derivative antibody thereof that binds said antigen for use in medicine.
- the invention provides a method of treating a patient in need thereof (e.g., a human patient), comprising administering a therapeutically effective amount of an isolated, monoclonal or polyclonal antibody described herein, or a mutant or derivative antibody thereof which binds an antigen.
- the invention provides a nucleotide sequence encoding an antibody described herein, optionally wherein the nucleotide sequence is part of a vector.
- the invention also provides a host cell comprising said nucleotide sequence.
- the invention provides a pharmaceutical composition comprising the antibody or antibodies described herein and a diluent, excipient or carrier.
- the invention provides an ES cell, a non-human animal or a non-human blastocyst comprising an expressible genomically-integrated nucleotide sequence encoding a Cas endonuclease (e.g., a Cas9 or Cys4) and optionally an expressible genomically-integrated nucleotide sequence encoding a tracrRNA or a gRNA.
- a Cas endonuclease e.g., a Cas9 or Cys4
- the ES cell is any ES cell type described herein.
- the endonuclease sequence is constitutively expressible.
- the endonuclease sequence is inducibly expressible.
- the endonuclease sequence is expressible in a tissue-specific manner in the animal or a progeny thereof, or in a non-human animal that is a progeny of the cell or blastocyst.
- the cell, animal or blastocyst comprises one or more gRNAs or an expressible nucleotide sequence encoding a gRNA or a plurality of expressible nucleotide sequences each encoding a different gRNA.
- the invention provides the use of the cell, animal or blastocyst in a method according to any configuration, aspect, embodiment or example of the invention.
- An aspect provides an antibody produced by the method of the invention, optionally for use in medicine, e.g., for treating and/or preventing (such as in a method of treating and/or preventing) a medical condition or disease in a patient, e.g., a human.
- nucleotide sequence encoding the antibody of the invention, optionally wherein the nucleotide sequence is part of a vector.
- Suitable vectors will be readily apparent to the skilled person, e.g., a conventional antibody expression vector comprising the nucleotide sequence together in operable linkage with one or more expression control elements.
- An aspect provides a pharmaceutical composition
- a pharmaceutical composition comprising the antibody of the invention and a diluent, excipient or carrier, optionally wherein the composition is contained in an intravenous (IV) container (e.g., and IV bag) or a container connected to an IV syringe.
- IV intravenous
- An aspect provides the use of the antibody of the invention in the manufacture of a medicament for the treatment and/or prophylaxis of a disease or condition in a patient, e.g. a human.
- the invention relates to humanised antibodies and antibody chains produced according to the present invention, both in chimaeric and fully humanised form, and use of said antibodies in medicine.
- the invention also relates to a pharmaceutical composition comprising such an antibody and a pharmaceutically acceptable carrier or other excipient.
- Antibody chains containing human sequences, such as chimaeric human-non human antibody chains, are considered humanised herein by virtue of the presence of the human protein coding regions region.
- Fully human antibodies may be produced starting from DNA encoding a chimaeric antibody chain of the invention using standard techniques.
- chimaeric antibodies or antibody chains generated in the present invention may be manipulated, suitably at the DNA level, to generate molecules with antibodylike properties or structure, such as a human variable region from a heavy or light chain absent a constant region, for example a domain antibody; or a human variable region with any constant region from either heavy or light chain from the same or different species; or a human variable region with a non-naturally occurring constant region; or human variable region together with any other fusion partner.
- the invention relates to all such chimaeric antibody derivatives derived from chimaeric antibodies identified according to the present invention.
- the invention relates to use of animals of the present invention in the analysis of the likely effects of drugs and vaccines in the context of a quasi-human antibody repertoire.
- the invention also relates to a method for identification or validation of a drug or vaccine, the method comprising delivering the vaccine or drug to a mammal of the invention and monitoring one or more of: the immune response, the safety profile; the effect on disease.
- the invention also relates to a kit comprising an antibody or antibody derivative as disclosed herein and either instructions for use of such antibody or a suitable laboratory reagent, such as a buffer, antibody detection reagent.
- a suitable laboratory reagent such as a buffer, antibody detection reagent.
- the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps
- A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
- A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
- expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
- the skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
- compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
- Mouse ES cells were transfected with human Cas9 nuclease and the two gRNAs. The transfection procedure was carried out as detailed above but the resulting clones were not selected. The transfected ES clones were genotyped using oligos pair spanning the two gRNA (Primer 1 & 2) to detect specific 55 bp deletion ( Figure 10).
- two gRNA or a single CRISPR array encoding multiple spacer sequence can be designed flanking a gene or a region of interest and with the association of Cas9 D10A nickase, two separate single-stranded breaks can be induced.
- This in association with a single-stranded DNA fragment containing DNA homology to the 5' breakpoint junction of the first DNA nick, and DNA homology to the 3' breakpoint junction of the second nick, the region in between the two single stranded DNA nick can be precisely deleted (Figure 2A).
- two separate gRNA or a multiplex single CRISPR array can be designed flanking a gene or a region of interest and with the association of Cas9 D10A nickase two separate single-stranded breaks can be induced.
- the intruding single stranded DNA fragment can contain DNA sequence from either endogenous or exogenous source containing sequence for a known gene, regulatory element promoter etc.
- This single-stranded DNA fragment (or double stranded DNA) can be brought together to replace the DNA region of interest flanked by DNA nick by arming it with DNA homology from the 5' region of the first nick and 3' region from the second nick ( Figure 3A).
- a selection marker can be included flanked by PiggyBac LTRs to allow for the direct selection of correctly modified clones. Once the correct clones have been identified, the selection marker can be removed conveniently through the expression of hyperactive piggyBac transposase (Yusa K., Zhou L, Li M.A., Bradley A., Craig N.L.: A hyperactive piggyBac transposase for mammalian applications., Proc. Natl. Acad. Sci. USA, 2011, 108(4): 1531-1536).
- the above approaches can be applied to ES cells, mammalian cells, yeast cells, bacterial cells, plant cells as well as directly performing in zygotes to expedite the process of homozygeous genome engineering in record time. It would be also possible to multiplex this system to generate multiple simultaneous DNA insertions (KI), deletions (KO) and the sequential deletion and insertion (KO - KI).
- gRNA single guide RNA
- a targeting vector was also constructed, which contained approximately 300 bp homology arms (5' and 3' HA) flanking the gRNA. The homology arms will hybridise exactly in the defined region and thus delete a 50 bp region, which is intended for deletion.
- the targeting vector also allows for the insertion of any DNA sequence of interest.
- the guide RNA (0.5 ug) together with the targeting vector (1 ug) and Cas9 nuclease vector (1 ug) was transfected into ES cells and 96 clones were picked after selection on puromycin using the protocol described above. Note. As a test for targeting efficiency, we compared linear verses circular targeting vector. Also as a negative control, we did the same experiment using no Cas9 vector to compare targeting efficiency via homologous recombination with and without Cas9 expression.
- Example 2 Recycling PAM for Sequential I nsertions or Delet ions
- the PAM sequence us recycled through reintroducing it via homologous recombination and as part of the homology arm.
- the PAM sequence can be optionally accompanied by a unique guide-RNA sequence creating a novel site within the host genome for further round of genome editing
- the CRISPR/Cas system can be used to rapidly and efficiently introduce lox sites or other recombinase recognition sequence such as Frt in a defined location to act as a landing pad for genome editing using recombinase mediated cassette exchange (RMCE) (Qiao 1, Oumard A., Wegloehner W., Bode J.: Novel tag-and-exchange (RMCE) strategies generate master cell clones with predictable and stable transgene expression properties., J. Mo/.
- RMCE recombinase mediated cassette exchange
- the RMCE step can be used to invert the region flanked by lox site or to delete this region as well as to simultaneously delete and insert DNA of interest in this region. Furthermore, the RMCE step can be adapted for carrying out multiple sequential rounds of RMCE (sRMCE).
- a piggyBac transposon harbouring a PGK promoter-driven loxP/mutant lox-flanked necf gene is targeted into an ES cell genome by standard homologous recombination.
- the targeted clones can be selected by G418. This provides a landing pad for the following recombinase-mediated cassette exchange (RMCE).
- RMCE recombinase-mediated cassette exchange
- Such an ES clone can be used a parental cells for any modification further.
- a cassette containing the loxP/mutant lox-flanked promoterless PurohTK-12K-Cas9 and U6 polymerase III promoter-driven guide RNA (gRNA) genes are inserted into the landing pad through transient ere expression.
- the gRNA genes can be one or more than one which target to the same gene or different genes.
- the inserted clones can be selected with puromycin and confirmed by junction PCRs. During the selection, the expression of Cas9 and gRNAs from the inserted cassette results in more efficient gene targeting or modification than transient expression of the Cas9 and gRNA can achieve. Following 4-6 day selection, the whole modified cassette is excised by the transient expression of piggyBac transposase (PBase). The final ES cell clones would not contain any Cas9 or gRNA sequence. The clones with homozygous modified genes would be confirmed by PCR and sequence.
- the main feature of this invention is to control the C sP and gRNA expression in certain time to be sufficient to generate efficient targeting rates.
- Targeting of the landing pad yielded many targeted ES clones.
- a selection of the targeted clones were used to insert a DNA cassette containing Cas9 nuclease linked to Puro- delta-tk via a T2A sequence into the targeted landing pad via RMCE, which involved the expression of Cre recombinase.
- the corresponding loxP and lo2272 sites within both the landing pad and the incoming vector ensured correct orientation of insertion. Since the landing pad contained a geneless PGK promoter, correct insertion of the incoming vector DNA containing Cas9, activated expression of puromycin and thus clones were positively selected on puromycin.
- Non-specific targeting of this DNA cassette will not yield puromycin resistant clones due to the absence of a promoter driving the transcription of the promoterless puromycin gene in the inserted DNA cassette.
- the initial Cas9 vector inserted into the landing pad did not contain any guide RNA sequence.
- the puromycin resistant ES clones were genotyped by PCR for the correct insertion of Cas9 ( Figure 12).
- the CRISPR/Cas genome editing system has been reconstructed in vitro and exemplified in mouse embryonic stem cells using vector pX330 containing humanised S, pyogenes i S Csnl) (Cong et a!.).
- the CRISPR/Cas system can be reconstructed as described in Cong et a! using synthetic DNA strings and DNA assembly.
- the entire DNA assembly would constitute a 6006 bp fragment containing 45 bp homology to pBlueScript S+ vector 5' to the EcoRV cutting site, Human U6 promoter, two Bbsl restriction sites for cloning in the spacer sequence which fuses to a chimeric guided RNA sequence, chicken beta-actin promoter with 3 FLAG, nuclear localisation signal (NLS) followed by hSpCsnl sequence and another NLS, bGH polyA, inverted terminal repeat sequence and finally another 45 bp homology to pBlueScript KS+ 3' to the EcoRV cutting site.
- This 6006 bp stretch of DNA will be synthetized as 7 individual DNA fragments where each fragment will have a 45 bp overlap to the adjacent DNA fragment to allow DNA assembly. The DNA sequence of these fragments is shown below in the order of assembly.
- Fragment 1 A ( 1 340 bp)
- the 6006 bp fragment can be assembled by assembly PCR by mixing molar ratio of the individual DNA fragments together and using the DNA mixture as PCR template.
- the assembled PCR product can then be cloned directly into pBlueScript vector or a standard cloning vector system such as a TOPO TA cloning kit (Invitrogen).
- the D10A nickase version of the CRISPR/Cas system can be conveniently reconstructed by assembling the above fragments where fragment 2 is replaced with fragment 2A which contains the D10A substitution (See sequence below).
- the target spacer sequence can be cloned into the above CRISPR/Cas vector system via the Bbsl restriction sites located upstream of the chimeric guided RNA sequence.
- the spacer sequence can be ordered as oligo pairs and annealed together with overhangs as shown below to allow direct cloning into Bbsl linearised CRISPR/Cas vector using standard molecular biology protocols.
- the 4 bp overhang sequence underlined is required to be included in the spacer oligos to facilitate cloning into the Bbsl restriction site in the CRISPR/Cas vector. Using this approach, any spacer sequence can be conveniently cloned into the CRISPR/Cas vector.
- the gRNA needs to be removed and synthetised separately by annealing oligos or produced synthetically (See below for an example T7-spacer sequence fused to chimeric guided RNA sequence - T7- gRNA).
- the spacer sequence will be designed in a unique region of a given chromosome to minimise off-target effect and also the respective protospacer genomic sequence needs to have a PAM at the 3'-end.
- fragment 1A containing 45 bp homology to pBlueScript KS+ vector 5' to the EcoRV restriction site, human U6 promoter, Bbsl restriction sites, chimeric guided RNA sequence and chicken b-actin promoter
- fragment 1A containing 45 bp homology to pBlueScript KS+ vector 5' to the EcoRV restriction site, human U6 promoter, Bbsl restriction sites, chimeric guided RNA sequence and chicken b-actin promoter
- fragment 1A containing 45 bp homology to pBlueScript KS+ vector 5' to the EcoRV restriction site, human U6 promoter, Bbsl restriction sites, chimeric guided RNA sequence and chicken b-actin promoter
- fragment 1A containing 45 bp homology to pBlueScript KS+ vector 5' to the EcoRV restriction site, human U6 promoter, Bbsl restriction sites, chimeric guided RNA sequence and chicken b-actin promoter
- fragment 1A containing 45 bp homo
- Fragment 1 ( 1 1 1 bp)
- DNA oligos ranging from 15 bp and upwards in excess of >125 bp will be synthetised through Sigma Custom Oligo synthesis Service.
- the oligos can contain any sequence such as a defined mutation, introduced restriction sites or a sequence of interest including recombination recognition sequence such as loxP or derivatives thereof, Frt and derivatives thereof or PiggyBac LTR or any other transposon elements or regulatory elements including enhancers, promoter sequence, reporter gene, selection markets and tags.
- the oligo design will incorporate DNA homology to the region where Cas9 mediates double-stranded DNA break or DNA nick. The size of the homology will range from a few base pairs (2-5 bp) to upwards and in excess of 80 bp.
- DNA fragments Larger DNA fragments (>100 bp ranging up to several kilobases) will be prepared either synthetically (GeneArt) or by PCR.
- the DNA fragment will be synthetised either with or without flanked NLS or only with a single NLS and either with or without a promoter (e,g, T7 polymerase promoter).
- the DNA can be prepared as a single stranded DNA fragment using either the capture biotinylated target DNA sequence method (Invitrogen: Dynabeads M-270 Streptavidin ) or any other standard and established single stranded DNA preparation methodology.
- the single stranded DNA can be prepared for microinjection by IVT as described above and the mRNA co- injected with Cas9 mRNA and gRNA.
- the DNA fragment can also be co-injected as a double stranded DNA fragment.
- the DNA fragment will be flanked by DNA homology to the site where Cas9 mediates double-stranded DNA break or DNA nick.
- the DNA homology can range from a few base pairs (2-5 bp) and up to or in excess of several kilobases.
- the DNA fragment can be used to introduce any endogenous or exogenous DNA.
- HDR-mediated repair can also be done in ES cells following CRISPR/Cas-mediated DNA cleavage.
- the above mentioned donor oligo or DNA fragment can be co-transfected into ES cells along with the CRISPR/Cas expression vector.
- Vector containing the T7 polymerase promoter with the coding region of humanised Cas9 will be PCR amplified using oligos Cas9-F and Cas9-R.
- the T7-Cas9 PCR product can be gel extracted and the DNA purified using Qiagen gel extraction kit.
- the purified T7-Cas9 DNA will be used for in vitro transcription (IVT) using m MESSAGE mMACHINE T7 Ultra Kit (Life Technologies Cat No. AM1345).
- the vector containing the T7-gRNA can be PCR amplified using oligos gRNA-F and gRNA-R and once again the PCR products gel purified.
- IVT of the T7-gRNA will be carried out using MEGAshortscript T7 Kit (Life Technologies Cat No. AM1354) and the gRNA purified using MEGAclear Kit (Life Technologies Cat No. AM1908) and eluted in RNase-free water.
- Cas9-F TTAATACGACTCACTATAGG (SEQ ID NO: 19)
- Cas9-R GCGAGCTCTAGGAATTCTTAC (SEQ ID NO: 20)
- gRNA-F TTAATACGACTCACTATAGG (SEQ ID NO:21)
- gRNA-R AAAAAAGCACCGACTCGGTGCCAC (SEQ ID NO:22)
- Example 5B One step generation of Transgenic animals
- Mouse embryonic stem cells AB2.1 and derivatives of this line will be used for transfecting the mammalian codon optimised Cas9 and sgRNA from a single expression vector or from separate vectors if desired.
- AB2.1 ES cells will be cultured on a PSNL76/7/4 MEF feeder layer in M-15: Knockout DMEM (Gibco, no pyruvate, high glucose, 15% FBS, lxGPS, lxBME) with standard ES cell culturing techniques.
- Transfection of CRISPR/Cas expression vector along with the optional addition of a donor oligo or DNA fragment will be done by electroporation using the Amaxa 4D-Nucleofector® Protocol (Lonza).
- a plasmid expressing PGK-Puro will also be optionally co-transfected to promote transfection efficiency.
- ES cells will be plated back onto feeder plates and Puromycin (2pg/ml) will be added 72 hours post transfection for 7 days after which colonies will be picked and genotyped by PCR. Positive colonies will be further cultured and expanded on feeder layer and selection markers where necessary will be excised using a PiggyBac transposon system. This will be done by electroporation of ES cells with a plasmid containing HyPbase using the Amaxa 4D-Nucleofector® Protocol (Lonza). The ES cell will be plated back onto feeder plates.
- ES cells will be passaged 2-3 days post transfection and after a further 2-3 days the ES cells will be plated out at different cells densities (1 :10, 1 :20, 1 :100 and 1 :300) and FIAU (2pg/ml) selection will be added 24 hours after replating. Colonies will be picked and analysed by PCR genotyping after 7-10 days on selection media. Positive clones will be further cultured and expanded on feeder layer and sent for zygote (blastocyst) microinjection. In an alternative method, 8 hours prior to transfection ES cells are seeded at a density of
- 0.5x106 cells using antibiotic free M-15 Knockout DMEM (Gibco, no pyruvate, high glucose, 15% FBS, lxL-Glutamine, lxBME) onto 6w feeder plates.
- Transient transfection is performed using Lipofectamine® LTX Reagent with PLUSTM Reagent (InvitrogenTM) by standard protocol. After incubation time transfection reagents are transferred to feeder plates (cultured in antibiotic free media), media (M-15) will not be changed on these plates for at least 24 hours post transfection. 48 hours post transfection ES cells are trypsinized into a single cell suspension and a cell count is carried out and cells are plated out at different cell densities ranging for 100-5000 cells per 10cm feeder plate.
- Method 5C Microinjection of Mouse Zygotes - Met hod 1 Materials and Reagents: . M2 (Sigma M7167)
- Zygotes are harvested from E0.5dpc (day post-coitum) superovulated female mice. The zygotes are incubated in hyaluronidase to disperse cumulus cells.
- Zygotes are collected and transferred to several drops of M2 medium to rinse off the hyaluronidase solution and debris. Zygotes are placed into KSOM Media and incubated at 37°C, 5% C0 2 until required.
- Zygote quality is assessed and zygotes with normal morphology are selected for injection, these are placed in KSOM media and incubated at 37°C, 5% C0 2 until required.
- Injection procedures are performed on a Nikon Eclipse Ti inverted microscope with Eppendorf micromanipulators and an Eppendorf femtojet injection system.
- a slide is prepared by adding a large drop ⁇ 200 microlitres of M2 into the centre.
- zygotes Place an appropriate number of zygotes onto the slide. Examine the zygotes and select only those with normal morphology (2 distinct pronuclei are visible). Whilst holding a zygote with a male pronucleus closest to the injection pipette, carefully push the injection pipette through the zona pellucida into the pronucleus, apply injection pressure, the pronucleus should visibly swell, remove the injection pipette quickly. The injected zygote can be placed down while the rest are injected.
- Day 0 Give PMSG (5 I.U.) to the females by I. P. injection. 2.
- Day 2 Give hCG (5 I.U.) to the females 48 Hours later by I. P. injection. Mate the females to stud males.
- Day 3 Check plugs, sacrifice plugged female mice by C02 asphyxiation or cervical dislocation at 0.5dpc at 8.00 am.
- the zygotes should be left in the hyaluronidase for a few minutes only, after which time the zygotes may become damaged. If necessary pipette them up and down a few times to help the release of the zygotes from the cumulus cells.
- Microinjection set up Injection procedures are performed on a Nikon Eclipse Ti inverted microscope with Eppendorf micromanipulators. Prepare a 60mm petri dish to place injected zygotes into. Pipette four - six 40 ⁇ drops of KSOM+AA, cover with oil and place in a 5% C0 2 incubator to equilibrate. Prepare a cavity slide by making a large ( ⁇ 200 ⁇ ) drop of M2 media onto the center of the well, add a small drop of medium on the left side of the slide, for the holding pipette.
- Microinjection Ensure that the pressurized injector has been switched on and is ready to use. Place an appropriate number of zygotes onto the slide, do not add more zygotes than can be injected within 20-30mins. Place the holding pipette into the drop of M2 on the left of the slide; it will fill using capillary action, once filled to about the shoulder attach to the manipulator. Carefully examine the zygotes, making sure that two pronuclei are visible and morphology is good, discard any that appear abnormal. To test if the injection needle is open, place the tip near to but not touching a zygote in the same focal plane.
- the zygote should be positioned in such a way that allows injection into the zygote without hitting the pronuclei, preferably with a gap between the zona pellucida and the oolema. Bring the tip of the injection needle into the same focal plane as the zona pellucida. Bring the injection pipette to the same y-axis position as the zona pellucida, adjust the height of the needle so the tip appears completely sharp, without changing the focus. This ensures the needle will target the zygote exactly. Push the injection pipette through the zona pellucida, through the cytoplasm towards the back of the zygote.
- the needle will create a "bubble" through the oolema, this needs to be broken, you will see it snap back at which point remove the needle quickly, you will see the cytoplasm moving to indicate RNA is flowing from the needle.
- Cytoplasmic granules flowing out of the oocytes after removal of the injection pipette is a clear sign that the zygote will soon lyse. In this case, or if nuclear/cytoplasmic components are sticking to the injection pipette, the oocytes should be discarded after injection. If the zygote appears to be intact and successfully injected, sort this into a good group. Pick a new zygote for injection. The same injection pipette can be used as long as it continues to inject successfully.
- mRNA from the guide RNA was also produced using in vitro transcription described above.
- oocytes were prepared from female mice using the protocol detailed above.
- An mRNA mixture containing lOOng/ul Cas9 nuclease mRNA and 50ng/ul guide mRNA was injected by microinjection into the cytoplasm as detailed above. The microinjection is done at the single-cell stage. Zygotes that survived the injection were cultured to 2 cell stage, which were then transferred to recipient mice.
- the male mouse (KMKY5.1c) that showed no WT sequence was used as a mating partner for the two female mice (KMKY5.1e & KMKY6.1e) that showed no WT sequence too.
- the resulting pups from the two matings yielded 14 pups in total from the first litter.
- Following similar sequencing analysis whereby PCR products amplified from the region around the guide RNA were cloned individual and several clones were then analysed for the presence of indels. For each mouse, 24 clones were analysed by sequencing. The sequencing data from all 14 pups confirmed only two indel sequences reflecting the two alleles arising from the parental male and female mouse.
- a landing pad consisting of a PiggyBac transposon element with the following features will be targeted into mouse ES cells (e.g., 129-derived ES cells, such as AB2.1 ES cells; Baylor College of Medicine, Texas, USA) and selected for on G418.
- the PiggyBac transposon element will contain neomycin resistance gene flanked by loxP and lox2272. It will also have a geneless PGK promoter.
- the landing pad will be targeted into the intragenic region of Rosa26 gene located on chromosome 6, but it could be targeted elsewhere.
- Targeting this landing pad in the Rosa26 gene will provide a universal ES cell line for precisely inserting any desired DNA fragment including DNA fragments containing Cas9, mutant Cas9 or any other gene of interest via RMCE with high efficiency.
- Targeting Rosa26 is beneficial since the targeted construct will be inserted as a single copy (unlike random integration elsewhere) and is unlikely to produce an unwanted phenotypic effect.
- This landing pad can be inserted into any gene in any chromosome or indeed in any eukaryotic or mammalian cell line, e.g., a human, insect, plant, yeast, mouse, rat, rabbit, rodent, pig, dog, cat, fish, chicken or bird cell line, followed by generation of the respective transgenic organism expressing Cas9.
- a human, insect, plant, yeast, mouse, rat, rabbit, rodent, pig, dog, cat, fish, chicken or bird cell line followed by generation of the respective transgenic organism expressing Cas9.
- Ubiquitous expression of transgene in mouse embryonic stem cell can be achieved by gene targeting to the ROSA26 locus (also known as: gene trap ROSA 26 or Gt(ROSA)26) by homologous recombination (Ref. (a) and (b) below).
- ROSA26 locus also known as: gene trap ROSA 26 or Gt(ROSA)26
- homologous recombination Repf. (a) and (b) below.
- the genomic coordinates for mouse C57BL/6J Rosa26 gene based on Ensemble release 73 - September 2013 is: Chromosome 6: 113,067,428 - 113,077,333; reverse strand.
- the Rosa26 locus can also be used to as a recipient location to knock-in a transgene.
- the Rosa26 locus can also be used to knock-in the landing pad vector by targeting through homologous recombination into the intronic region located between exons 2 and 3 of mouse strain 129-derived embryonic stem cells using approx. 3.1 kb homology arms.
- the homology arms were retrieved by recombineering from a BAC Clone generated from mouse strain 129.
- the sequence of the Rosa26 homology arms used for targeting is given below.
- a recombinase mediated cassette exchange (RMCE)-enabled vector containing a promoterless puromycin-delta-tk with in-frame fusion of T2A at the C-terminus following by either Cas9 or mutant Cas9 nucleotide sequence and a series of unique restriction sites flanked by loxP and lox2272 will allow for the direct targeting of this vector into the landing pad by Cre-mediated RMCE.
- T2A allows ribosomal skipping during translation. The insertion of the coding sequence of T2A between two genes results in two products (one gene, one transcript but two proteins expressed, in this case the Cas9 and selection marker).
- ES clones containing the correctly inserted DNA fragment can be directly selected on puromycin.
- This approach also advantageously ensures single copy expression of Cas9 as suppose to a random integration or transient expression approach. Insertion of the RMCE enabled vector into the desired locus containing the landing pad can be selected directly as the PGK promoter in the landing pad will drive the transcription of the promoterless Puro- Delta-Tk and Cas9. Since the Puro-delta-Tk is in the same transcriptional unit as Cas9, ES clones selected on puromycin will ensure expression of Cas9.
- the above strategy allows for three separate approaches to express the sgRNA designed for disrupting (mutation through indel formation, deletion or deletion followed by insertion) gene of interest.
- the above ES cell line containing Cas9 can be used for generating transgenic mice with either constitutively expressed Cas9 or modified for inducible Cas9 expression or indeed tissue specific Cas9 expression for example expression of Cas9 at an embryo stage using Nanog-, Pou5fl- or SoxB promoter-specifc Cas9 expression.
- Such derived mouse line expressing Cas9 can be used for genome editing in a streamline fashion whereby in vitro transcribed sgRNA can be easily injected into embryos obtained from such transgenic mice. This will enhance the efficiency of generating mouse lines with the desired homozygous genotype and thus will dramatically reduce the number of animals required.
- sgRNA can be transfected directly into the ES cells expressing Cas9 and thus avoids the requirement for cloning into the RMCE enabled vector single or multiple sgRNA. This approach will allow multiple sgRNA to be inserted into the ES cells simultaneously very rapidly.
- sgRNA can be cloned directly into the multiple cloning site of the RMCE enabled vector (ie, using a plurality of different restriction endonuclease sites) to allow single copy expression of the guide-RNA. This approach may be useful for limiting off-target effects particularly relevant for those genes with high sequence homology within the genome.
- ES cells expressing Cas9 and sgRNA can be selected for directly on medium containing puromycin. Selection on puromycin for 4-6 days will allow for the desired location to be mutated or disrupted and the advantage of manipulating ES cells is that individual clones can be analysed by PCR followed by sequencing for the desired mutation. Only correctly mutated ES cell clones can be processed further whereby inserted DNA element introduced through insertion of the landing pad and the subsequent insertion of the RMCE vector can be completely removed leaving the ES cell devoid of any alteration other than the intended mutation induced by the action of Cas9 and the sgRNA. This can be done through transiently expressing PBase transposon followed by selection on FIAU. Removal of the constitutively expressed Cas9 with only the minimal length of time required to induce mutation in the presence of sgRNA will reduce or eliminate the possibility of Cas9 inducing unwanted mutations.
- ES Clones containing the desired mutation can be injected into blastocyst to generate transgenic mice.
- Genomes are abbreviated according to the denominations of the species or genera carrying the corresponding CRISPR arrays: M , M. t erm utotrophicus; Lmo, !. monocytogenes ; Eco, K coli; Pae, P. aeruginosa; Spy, S. pyogenes; Xan, Xanthomonas spp.; She, Shewanella spp. ; Ype, Y. pestis; Sso, S. solfataricus ; se, M. sedula; Str,
- Streptococcus spp. Lis, Listeria spp.
- NC_008601.1 810052..814941
- NC_002163.1 (1456880..1459834, complement)
- NC_010816.1 (2257993..2261556)
- NC_004829.2 (919248..923060)
- NC_009257.1 (1332426..1335803, complement) ID: 4958852
- NC_016749.1 (1418337..1421729, complement)
- NC_015433.1 (1323872..1327240, complement)
- NC_013798.1 (1511433..1514825, complement)
- NC_013798.1 (1518984..1523110, complement)
- NC_013714.1 (1400576..1403992, complement)
- NC_013016.1 (369547.372795, complement)
- NC_011134.1 (1369339..1373385, complement)
- NC_009785.1 (1426750..1430160, complement)
- cytoplasmic protein [Streptococcus pyogenes MGAS6180]
- NC_015215.1 (1520905..1525017, complement)
- NC_002967.9 (361021..365208)
- NC_015600.1 1400035..1403427, complement
- CRISPR-associated protein [Corynebacterium ulcerans BR-AD22]
- NC_015683.1 (30419..33112, complement)
- cytoplasmic protein [Streptococcus pyogenes MGAS9429]
- NC_012781.1 (1591112..1594456)
- NC_008532.1 (1379975..1384141, complement)
- NC_008532.1 (643235..646600)
- NC_015389.1 (2036091..2040245)
- NC_015321.1 (3610221..3614597, complement)
- NC_015138.1 (295839.-298976)
- NAL21 2 2952 CRISPR-associated protein, Csnl family [Nitrosomonas sp. AL212] Other Aliases: NAL212_2952
- NC_015152.1 (2367952..2371491, complement)
- NC_011365.1 (382737.385748)
- NC_009655.1 431928..435116
- NC_008786.1 (1365979..1369185)
- NC_008022.1 (844446.-848552)
- NC_004368.1 (945801..949946)
- NC_015846.1 (1579873..1584165, complement) ID: 10980451
- NC_017503.1 (888602..892411)
- NC_013515.1 (1159048..1162827, complement)
- NC_009839.1 (1442672..1445626, complement) ID: 5618449
- NC_018712.1 (1169559..1173674, complement)
- NC_018089.1 1320641..1324678, complement
- NC_017927.1 (1376653..1380819, complement)
- NC_017927.1 (624274.-627639)
- NC_017569.1 (1443996..1448198)
- STND 0658 CRISPR-associated endonuclease, Csnl family [Streptococcus thermophilus ND03]
- NC_017563.1 633621..636986
- NC_017045.1 (1023494..1026931, complement)
- NC_016826.1 (1276484..1280611, complement)
- NC_014935.1 (1477331..1480729)
- NC_013928.1 (737258..741295)
- Chromosome 1NC_012891.1 (1176755..1180870, complement) ID: 8111553
- NC_010120.1 (402733..405981, complement)
- Genomic context Chromosome Annotation: NC_007297.1 (773340..777446)
- NC_007294.1 (684155..688099)
- NC_005090.1 (1525628..1529857)
- NC_002663.1 1324015..1327185, complement
- SPs1 1 hypothetical protein [Streptococcus pyogenes SSI-1]
- NC_004350.2 (1330942..1334979, complement)
- NC_003212.1 (2770707..2774711, complement)
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Abstract
L'invention concerne une approche pour introduire une ou plusieurs insertions et/ou délétions désirées de taille connue dans un ou plusieurs sites prédéfinis dans un acide nucléique (par exemple dans un génome de cellule ou d'organisme). Des techniques ont été développées pour y parvenir de manière séquentielle ou par insertion d'un fragment d'ADN discret de taille définie dans le génome précisément dans un site prédéfini ou pour effectuer une délétion discrète d'une taille définie dans un site précis. La technique repose sur l'observation selon laquelle des ruptures monocaténaires d'ADN sont préférentiellement réparées par la voie HDR, et cela réduit les risques d'indels (par exemple produites par des jonctions d'extrémités non homologues, NHEJ) dans la présente invention et est donc plus efficace que les techniques de l'art antérieur. L'invention concerne également des insertions et/ou délétions séquentielles au moyen de coupure d'ADN mono- ou bicaténaire.
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EP14772198.9A EP2877571B1 (fr) | 2013-09-18 | 2014-09-18 | Procédés, cellules et organismes |
EP20212097.8A EP3842528A1 (fr) | 2013-09-18 | 2014-09-18 | Procédés, cellules et organismes |
EP18174860.9A EP3418379B1 (fr) | 2013-09-18 | 2014-09-18 | Procédés, cellules et organismes |
CN201480051543.4A CN105637087A (zh) | 2013-09-18 | 2014-09-18 | 方法、细胞与生物体 |
ES14772198.9T ES2681622T3 (es) | 2013-09-18 | 2014-09-18 | Métodos, células y organismos |
EP21196419.2A EP3988649A1 (fr) | 2013-09-18 | 2014-09-18 | Procédés, cellules et organismes |
US15/072,978 US20160257974A1 (en) | 2013-09-18 | 2016-03-17 | Methods, Cells & Organisms |
US15/072,794 US20160257948A1 (en) | 2013-09-18 | 2016-03-17 | Methods, Cells & Organisms |
US15/610,384 US20170275611A1 (en) | 2013-09-18 | 2017-05-31 | Methods, cells & organisms |
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Cited By (57)
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WO2015159087A1 (fr) * | 2014-04-17 | 2015-10-22 | Green Biologics Limited | Mutations ciblees |
US9228207B2 (en) | 2013-09-06 | 2016-01-05 | President And Fellows Of Harvard College | Switchable gRNAs comprising aptamers |
US9228208B2 (en) | 2013-12-11 | 2016-01-05 | Regeneron Pharmaceuticals, Inc. | Methods and compositions for the targeted modification of a genome |
US9322006B2 (en) | 2011-07-22 | 2016-04-26 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
US9359599B2 (en) | 2013-08-22 | 2016-06-07 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
US9388430B2 (en) | 2013-09-06 | 2016-07-12 | President And Fellows Of Harvard College | Cas9-recombinase fusion proteins and uses thereof |
US9526784B2 (en) | 2013-09-06 | 2016-12-27 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
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2014
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- 2014-09-18 EP EP18174860.9A patent/EP3418379B1/fr not_active Revoked
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- 2014-09-18 ES ES14772198.9T patent/ES2681622T3/es active Active
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2016
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2017
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US20170275611A1 (en) | 2017-09-28 |
EP3418379A2 (fr) | 2018-12-26 |
US20150079680A1 (en) | 2015-03-19 |
US20160257948A1 (en) | 2016-09-08 |
ES2681622T3 (es) | 2018-09-14 |
CN105637087A (zh) | 2016-06-01 |
US20160207983A1 (en) | 2016-07-21 |
TW201542816A (zh) | 2015-11-16 |
EP3418379A3 (fr) | 2019-02-13 |
ES2844174T3 (es) | 2021-07-21 |
EP3842528A1 (fr) | 2021-06-30 |
US20190338274A1 (en) | 2019-11-07 |
US11920128B2 (en) | 2024-03-05 |
US20160257974A1 (en) | 2016-09-08 |
US20160177340A1 (en) | 2016-06-23 |
EP3988649A1 (fr) | 2022-04-27 |
EP2877571B1 (fr) | 2018-05-30 |
EP2877571A1 (fr) | 2015-06-03 |
EP3418379B1 (fr) | 2020-12-09 |
DE202014010413U1 (de) | 2015-12-08 |
US20240200054A1 (en) | 2024-06-20 |
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